The enzyme linked detection of binding reactions on porous substrates is well known in the art. For example, U.S. Pat. No. 4,452,901 (Gordon) discusses a method for transferring electrophoretically separated protein molecules from a gel to a porous nitrocellulose sheet, while preserving an exact replica of the electrophoretic separation. The separated, immobilized proteins can then be specifically probed, or further studied using antibodies. In order to localize the reaction on the membrane, however, the enzyme product must remain in the vicinity of the reaction. In many cases, this may be accomplished by the formation of an insoluble product which falls to the surface of the membrane around the reaction site. More recently, enzyme substrates yielding unstable products which further decay by emitting light have been tested (Pollard-Knight et al., Analytical Biochemistry 185, 353-358, 1990; U.S. Pat. No. 4,857,652). Since the unstable products are short-lived, they do not have time to diffuse far from the site of the reaction before light is emitted. The light signal may be captured on film.
In any enzyme reaction the rate of product formation is dependent on several factors. One of the most important is the concentration of the substrate available to the enzyme. For product formation to proceed at maximum rate, the concentration of the substrate must exceed the Km of the enzyme. If an enzyme reaction occurs in solution, the substrate which gets consumed by the reaction is rapidly replaced by new substrate contained in the solution. Where the enzyme is in an environment having a barrier to diffusion, however, the local concentration of substrate may decline as the reaction proceeds. This may eventually cause a decline in the rate of product formation. Maintaining high substrate concentration is therefore important to maximize the rate of product formation.
Normally, with membrane based enzyme linked reactions, the membrane is submersed in a solution of enzyme substrate which acts as a constant reservoir of substrate. In general this prevents the concentration of substrate from becoming too low or rate limiting, and allows the enzyme reaction to proceed as rapidly as possible. This method requires excess substrate, and the reaction continues for a long period of time. During that time, the measurement of product formation remains difficult. Some patentees have attempted to separate substrate from other reactants by constructing multizone devices where enzyme substrates are immobilized in a layer into which the reactants diffuse (see e.g., U.S. Pat. Nos. 4,806,312, 4,959,305 4,975,366). However, such devices contain the enzyme product within a particular location, and the enzyme substrate is not freely diffusible to the reaction site.
In the example of chemiluminescent substrates, such as the derivatives of the 1,2-dioxetanes, which can be used to detect a membrane-based reaction by exposing on film, an adamantyl derivative confers sufficient stability to the dioxetane to allow its use. Lumigen PPD (Lumigen, Inc. Detroit, Mich.), a form of dioxetane which can be dephosphorylated by alkaline phosphatase, generates an unstable phenolate anion AMP-D which eventually emits light. AMP-D, even though it has a half-life on the order of minutes, does not diffuse far from the site of enzymatic activity. In addition, the quantum yield of light from the dioxetane derivative is dramatically enhanced in this type of environment. The lack of diffusion is important because it allows the preservation of the resolution originally gained by the gel. Thus these dioxetane derivatives have been shown to be useful for detection of Western blots by accumulating the light signal on film.
In normal application, a membrane containing an enzyme-linked binding assay is submersed in a solution of substrate (dioxetane) and appropriate buffers and salts. Excess reagent is then drained from the blot, the membrane wrapped in plastic and exposed to X-ray film for varying periods of time. Following exposure of the blot to the dioxetane substrate there is a gradual increase in light production from those regions of the blot which contain enzyme. The rate of light production increases over a period of hours. When the maximum rate of light production has been reached the detection system is most sensitive. Thus, improvements in speed or sensitivity as well as improvements which use less substrate would be advantageous.